Mechanism of Translation Termination: RF1 Dissociation Follows Dissociation of RF3 from the Ribosome

Release factors 1 and 2 (RF1 and RF2, respectively) bind to ribosomes that have a stop codon in the A site and catalyze the release of the newly synthesized protein. Following peptide release, the dissociation of RF1 and RF2 from the ribosome is accelerated by release factor 3 (RF3). The mechanism for RF3-promoted dissociation of RF1 and RF2 is unclear. It was previously proposed that RF3 hydrolyzes GTP and dissociates from the ribosome after RF1 dissociation. Here we monitored directly the dissociation kinetics of RF1 and RF3 using Förster resonance energy transfer-based assays. In contrast to the previous model, our data show that RF3 hydrolyzes GTP and dissociates from the ribosome before RF1 dissociation. We propose that RF3 stabilizes the ratcheted state of the ribosome, which consequently accelerates the dissociation of RF1 and RF2

Structure of RF1 bound to the ribosome.

<p>(A) RF1 (green) bound to the ribosome (grey) in the ribosomal A site with P site tRNA (purple), E site tRNA (orange), and mRNA (pink). (B) Detailed view on the decoding site showing RF1 residues (green), base G530 of 16S rRNA (grey) and the stop codon UAA (pink). The structure figures were prepared from PDB file 3D5A using PyMol. (C) and (D) Close up of the interactions between the stop codon (pink) and the RF1 residues (green). <i>E. coli</i> numbering is used for RF1 residues. Hydrogen bonds are indicated by the dotted lines.</p

Kinetics of wild type RF1 and RF1 mutants binding to the ribosome.

<p>Representative stopped-flow time course of 1 μM wild type RF1 (A) and 1 μM RF1 mutants Q185A (B), R186A (C), T190A (D), and T198A (E) binding to ribosome. The time courses (grey trace) were transformed and fit to a double-exponential equation (black line) to determine the observed rates of RF1 binding (<i>k</i>_obs1 and <i>k</i>_obs2).</p

Fluorescence assay for determining the K_D of RF1 binding to the ribosome.

<p>(A) Changes in relative fluorescence intensity after adding increasing concentrations of wild type RF1 (open diamonds) and RF1 mutants Q185A (filled circles), R186A (filled triangles), T190A (open circles), and T198A (open squares) to ribosomes programmed with a UAA stop codon. (B) Changes in relative fluorescence intensity after adding increasing concentrations of wild type RF1 (open diamonds), and RF1 mutants Q185A (filled circles), R186A (filled triangles), T190A (open circles), and T198A (open squares) to ribosomes programmed with a UGA stop codon. Representative titration experiments without standard deviations are shown and the data were fit to the quadratic equation (black line). The total RF1 concentrations added are indicated on the x-axis.</p

Concentration dependence of the observed rate of RF1 binding.

<p>(A) Concentration dependence of the observed rate for phase 1 of RF1 binding. Plots were fit to a linear equation to determine the association (<i>k</i>₁) and dissociation (<i>k</i>₋₁) rate constants. (B) Concentration dependence of the observed rate for phase 2 of RF1 binding. Plots were fit to a linear equation. The standard errors from three independent experiments are shown. Indicated are wild type RF1 (open diamonds), RF1 mutants Q185A (filled circles), R186A (filled triangles), T190A (open circles), and T198A (open squares).</p

Kinetics of peptide hydrolysis by RF1.

<p>(A) Representative TLC displaying the time course of RF1-catalyzed release of [³⁵S]-fMet from ribosome release complex. Labels indicate wild type RF1 and RF1 mutants. The final extent of peptide release by wild type and RF1 mutants were similar and separate filter binding studies showed that the extent of peptide release by wild type RF1 with UAA codon was >90%. (B) Graph showing the peptide release time course at saturating concentrations of wild type RF1 (open diamonds), RF1 mutants Q185A (filled circles), R186A (filled triangles), T190A (open circles), and T198A (open squares) are shown. Data were individually normalized and fit to a single-exponential equation (black line) to determine the rate of peptide release. Standard errors from at least three independent experiments are shown.</p

Thermodynamics and kinetics of RF1 mutants binding to the ribosome.

<p>*taken from 17.</p

RNA Modulates the Interaction between Influenza A Virus NS1 and Human PABP1

Nonstructural protein 1 (NS1) is a multifunctional protein involved in preventing host–interferon response in influenza A virus (IAV). Previous studies have indicated that NS1 also stimulates the translation of viral mRNA by binding to conserved sequences in the viral 5′-UTR. Additionally, NS1 binds to poly­(A) binding protein 1 (PABP1) and eukaryotic initiation factor 4G (eIF4G). The interaction of NS1 with the viral 5′-UTR, PABP1, and eIF4G has been suggested to specifically enhance the translation of viral mRNAs. In contrast, we report that NS1 does not directly bind to sequences in the viral 5′-UTR, indicating that NS1 is not responsible for providing the specificity to stimulate viral mRNA translation. We also monitored the interaction of NS1 with PABP1 using a new, quantitative FRET assay. Our data show that NS1 binds to PABP1 with high affinity; however, the binding of double-stranded RNA (dsRNA) to NS1 weakens the binding of NS1 to PABP1. Correspondingly, the binding of PABP1 to NS1 weakens the binding of NS1 to double-stranded RNA (dsRNA). In contrast, the affinity of PABP1 for binding to poly­(A) RNA is not significantly changed by NS1. We propose that the modulation of NS1·PABP1 interaction by dsRNA may be important for the viral cycle